# load necessary libraries
Warning message:
R graphics engine version 15 is not supported by this version of RStudio. The Plots tab will be disabled until a newer version of RStudio is installed.
library(Seurat)
Registered S3 method overwritten by 'data.table':
method from
print.data.table
Registered S3 method overwritten by 'htmlwidgets':
method from
print.htmlwidget tools:rstudio
Attaching SeuratObject
library(dplyr)
Attaching package: ‘dplyr’
The following objects are masked from ‘package:stats’:
filter, lag
The following objects are masked from ‘package:base’:
intersect, setdiff, setequal, union
# library(CelltypeR)
Read in the flow data This data should be the gated live cells.
All samples need to be in one folder.
input_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/FlowDataFiles/9MBO"
output_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary"
# 1.a Read in FlowJo Files
# choose to downsample to 9000 where possible
# check this - I don't think it worked
flowset <- fsc_to_fs(input_path, downsample = 9000)
# down sample can be a number, 'none' or 'min'
# look at file names and rename with shorter sample names
sampleNames(flowset)
sampleNames(flowset) <- sampleNames(flowset) <- c("3450_0306","AIW002_0306","AJG001C_0306","3450_0317A","AIW002_0317A","AJG001C_0317A","3450_0317B","AIW002_0317B","AJG001C_0317B")
sampleNames(flowset)
# if we want to save the flowset object now we can
# this would be read back in with flowset
#
Harmonize data to remove batch or technical variation
This requires us to look and see where there are two peaks to align. We need to visualize the peaks of the transformed data before aligning.
# we can decided what level of processing to choose with the argument 'processing'
# biexp only applies a biexponential transformation
# align applies biexp transform and then aligns peaks
# retro (default), transforms, aligns and then reverse transforms
flowset_biexp <- harmonize(flowset, processing = 'biexp')
# we can visualize the peaks to see where there are two peaks for alignment
# we need to enter the column index for which peaks to align, the alignment for one or two peaks is not the same.
#plotdensity_flowset(flowset)
plotdensity_flowset(flowset_biexp) # to see the peaks
flowset_align <- harmonize(flowset, processing = 'align',
two_peaks = c(7:20),
one_peak = c(1:6,21), threshold = 0.01)
flowset_retro <- harmonize(flowset, processing = 'retro',
two_peaks = c(7:20),
one_peak = c(1:6,21), threshold = 0.01)
df <- flowset_to_csv(flowset_retro)
Now we have made all the different processing of the fsc files. We can visualize the intensity in cell density plots to see the alignment
#plotdensity_flowset(flowset)
plotdensity_flowset(flowset_biexp)
plotdensity_flowset(flowset_align)
#plotdensity_flowset(flowset_retro)
Now we will make test out clustering. For Seurat clustering and Phenograph we will make the Seurat object Flowsome takes in the dataframe directly.
Save dataframe and seurat object for later
output_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary/"
# to save the df for later
# write.csv(df, "pathway/filename.csv")
write.csv(df, paste(output_path,"df9000.csv", sep = ""))
# save the seurat object
saveRDS(seu, paste(output_path,"seu9000.RDS", sep = ""))
Start from saved preprocessing
in_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary/"
df <- read.csv(paste(in_path,"df9000.csv", sep = ""))
seu <- readRDS(paste(in_path,"seu9000.RDS", sep = ""))
Annotate clusters 1. Visualization for manual annotation. - output by clustering function 2. CAM (Correlation assignment model) - requires reference matrix 3. RFM (Random Forest Model) - requires annotated matching flow dataset 4. Seurat label transfer - requires annotated matching flow data in a seurat object
Visualize expression on UMAP and with heat maps
output_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary/"
saveRDS(seu, paste(output_path,"cluster9000.RDS"))
Predict cell annotations with CAM (Corralations assignment method)
# read in the clustered unlabeled Seurat object and the expression data frame (for correlation)
output_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary/annotate/"
#seu <- readRDS("/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary/cluster9000.RDS")
#create the output pathway
test_path <- "/Users/rhalenathomas/Documents/Data/FlowCytometry/PhenoID/Analysis/testingLibrary/df9000.csv"
reference_path <- "/Users/rhalenathomas/GITHUB/PhenoID_single_cell_flow_cytometry_analysis/old/correlation/ReferenceMatrix9celltypesOrdered.csv"
test_data <- read.csv(test_path)
# dataframe output from fsc_df
reference_data <- read.csv(reference_path)
cor <- find_correlation(test_data,
reference_data,
min_corr = 0.45,
min_diff = 0.05)
class(cor)
[1] "data.frame"
# creates a dataframe with cor1 cor2 and predicted cell type label
Visualize the CAM results
plot_corr(cor)
Using X, best.cell.type, second.cell.type, cell.label as id variables
Using X, best.cell.type, second.cell.type, cell.label as id variables
Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
ℹ Please use `linewidth` instead.
This warning is displayed once every 8 hours.
Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated.
Using X, best.cell.type, second.cell.type, cell.label as id variables
[[1]]
[[2]]
[[3]]
[[4]]
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[[7]]
Warning: Removed 35 rows containing missing values (`geom_line()`).
Warning: Removed 35 rows containing missing values (`geom_point()`).
Apply correlation predictions to clusters and output a vector for annotation functions
Run get annotations function to return a vector of annotation in the order of the clusters.
# the output of this function
#get_annotation(seu, cluster, lables, top_n = 3, ignore_unknown = FALSE)
# get annotation predicted for each cluster
# a function to get a dataframe for annotation
# takes in a seurat object with clusters and predicted labels
# takes the cluster meta data slot, the predicted labels slot
# take number of different top predictions per cluster to view
# later add in option if > X% of cells are unknown keep unknown
# will return a dataframe with label for each cluster number
cor.ann <- get_annotation(seu.t, seu.t$RNA_snn_res.0.8,seu.t$cor.labels,
top_n = 3, ignore_unknown = TRUE, Label = "CAM")
[1] "Annotations in order of clusters starting at 0"
cor.ann
NA
NA
Use a trained Random Forest model to predict cell types. Training of the Random Forest model with an annotated data set is below.
table(seu.t$rfm.labels)
Unknown Mixed Neurons 1 Radial Glia 1
20 54531 9322 131
Epithelial Astrocytes 1 Neurons 2 Astrocytes 2
5154 192 0 1014
Astrocytes 3 Astrocytes mature Neurons 3 NPC
63 494 1801 504
Radial Glia 2 Radial Glia 3 Endothelial Oligodendrocytes
29 5 318 0
Stem-like 1 Stem-like 2 Neural stem
0 0 0
Get the annotation by cluster for the RFM
rfm.ann <- get_annotation(seu.t, seu.t$RNA_snn_res.0.8,seu.t$rfm.labels,
top_n = 3, ignore_unknown = TRUE, Label = "RFM")
[1] "Annotations in order of clusters starting at 0"
rfm.ann
NA
NA
Plot RFM predictions
plot_lab_clust(seu.t, seu.t$RNA_snn_res.0.8, seu.t$rfm.labels)
NA
NA
Predicting cell types wiht Seurat label transfer using anchors
seu.2 <- seurat_predict(seu.q, seu.r, ref_id = 'subgroups', down.sample = 500, markers = AB)
Projecting cell embeddings
Finding neighborhoods
Finding anchors
Found 35142 anchors
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Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Warning: Requested number is larger than the number of available items (13). Setting to 13.
Filtering anchors
Retained 17685 anchors
Finding integration vectors
Finding integration vector weights
0% 10 20 30 40 50 60 70 80 90 100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Predicting cell labels
seu.ann <- get_annotation(seu.2, seu.2$RNA_snn_res.0.8,seu.2$seu.pred,
top_n = 3, ignore_unknown = TRUE, Label = "Seurat")
[1] "Annotations in order of clusters starting at 0"
seu.ann
Get a consensus of cluster annotations, Add the annotations to the seurat object
seu <- cluster_annotate(seu, ann.list,
annotation_name ="CellType3",
to_label = "RNA_snn_res.0.8")
Train Random Forest model Requires a labelled seurat object More cells will give higher accuracy but increase computation time. Run in HPC with lots of cells
# Defaults
# RFM_train(seurate_object, AB_list, annotations = seu$CellTypes,split = c(0.5,0.5), downsample = 'none',seed = 222, mytry = c(1:10), maxnodes = c(12: 25), trees = c(250, 500, 1000,2000), start_node = 15)
rf <- RFM_train(seurate_object = seu,
AB_list = AB, annotations = seu$CellTypes,
split = c(0.8,0.2),
downsample = 5000,
seed = 222,
mytry = c(1:10),
maxnodes = c(12: 25),
trees = c(250, 500, 1000,2000),
start_node = 15)
save(rf, output_path,"trainedRFM.Rds")